Our laboratory recently discovered that microsomal triglyceride transfer protein (MTP), a protein essential for the assembly of triglyceride-rich lipoproteins within hepatocytes and enterocytes, is expressed in adipocytes of mice, rats, and humans. In addition, MTP is expressed in 3T3-L1 cells, and expression increases ~5-fold when these cells are induced to differentiate into adipocytes. MTP is present in a punctate pattern on the surface of lipid droplets and is especially prominent at the points of contact of adjacent lipid droplets. When MTP expression is silenced and the cells induced to differentiate, only very small (<1 5m) lipid droplets are formed. In addition, when MTP is transfected into CHO cells and lipid droplet formation stimulated, larger droplets are formed compared to those formed in cells without MTP. Finally, knocking down MTP expression in mouse adipose tissue using Cre-Lox approaches leads to decreased adiposity and a leaner mouse than littermate controls. Our working hypothesis is: MTP is a critical component in the maturation and expansion of lipid droplets within the adipocyte. This working hypothesis will be tested as follows: Specific Aim 1: To define the requirement for MTP in lipid droplet maturation. These studies will test the hypotheses that MTP facilitates normal lipid droplet expansion and growth and that the absence of MTP in adipocytes in vivo results in leaner mice with reduced body fat and improved glucose metabolism. Using primary pre-adipocytes isolated from fat specific MTP-knockdown mice and 3T3-L1 cells, in which MTP expression has been silenced by siRNA, we will evaluate the effects of MTP deficiency on differentiation, lipid droplet formation and maturation, de novo lipogenesis, and glycerol metabolism. We will also explore the effects of enhanced MTP expression on lipid droplet formation and maturation in primary pre-adipocytes and 3T3 cells. Using fat-specific MTP knockdown mice, we will define the effects of the knockdown on body weight, body fat composition, energy expenditure, food consumption, leptin, and insulin. Glucose metabolism will also be assessed using glucose tolerance tests as well as hyperinsulinemic euglycemic clamps. Specific Aim 2. To determine the molecular mechanism by which MTP facilitates the growth and maturation of lipid droplets. These studies will test the hypothesis that MTP facilitates lipid droplet expansion via structural and functional properties of the molecule. Selected sites in the putative lipid binding region of MTP will be mutated to determine if MTP functions as a "fusogenic protein" in lipid droplet maturation. MTP inhibitors will be used to determine if lipid transfer activity is necessary for lipid droplet expansion. Specific Aim 3. To examine the mechanism by which MTP associates with lipid droplets in 3T3- L1 cells. These studies will test the hypothesis that association of MTP with triglyceride in specific regions in the ER membrane initiates the movement of MTP from the lumen of the ER to the lipid droplet surface. Confocal and electron microscopy as well as biochemical approaches will be used to examine subcellular locations of MTP in differentiating 3T3-L1 cells and its association with lipid droplets in comparison with selected members of the PAT family of lipid droplet proteins. PUBLIC HEALTH RELEVANCE: Obesity presents a major health problem in the United States, even among American Veterans. A study of Veterans receiving care at VA medical facilities during 2000, revealed that 68.4% of the women were overweight (BMI 25) with 37.4% classified as obese (BMI 30). Seventy-three percent of the men were overweight with 32.9% classified as obese. This epidemic poses a major health problem as obesity is associated with a number of complications include type 2 diabetes, insulin resistance, dyslipidemia, hypertension, and cardiovascular disease. Unfortunately, little is known about the pathophysiological basis of obesity, and the mechanisms that underlie the excessive accumulation of fat. Indeed, little is known about the basic steps in lipid droplet formation and maturation within the fat cell and the mechanisms that regulate fat deposition. The proposed studies will provide new insight into how fat is stored and could prove important in understanding the factors involved in obesity and its many complications, including cardiovascular disease.